The present invention relates to biolelectronic devices comprising living cells which are in operative contact with an extracellular planar potential-sensitive electrode, e.g. a field effect transistor. The cells comprise an ion channel/receptor system which is responsive to stimuli. Thus, the device is suitable as a bioelectronic sensor. The electode may also have a capacitive stimulating spot, with which the electrical or functional state of the cell or its ion channel/receptor system may be affected.
To determine the pharmaceutical effect of test substances, often so-called cellular screening assays are performed in which a cell to be tested containing a receptor system is brought into contact with a test substance in order to examine its function as an effector on the cellular receptor system. These test procedures are often complicated and expensive. Thus, there is a need for devices and methods which allow a quick and efficient screening of many cells. These could simplify the procedure of pharmaceutical tests.
According to the present invention, this problem is solved by combining receptor-effector systems with the functional characteristics of ion channels. The activity of these ion channels is modulated due to the effect of the receptor-effector system. This modulation can be detected by an extracellular planar potential sensitive electrode.
Thus, a subject matter of the present invention is bioelectronic device comprising
a) a cell which expresses an ion channel/receptor system wherein said ion channel is responsive to a change in the functional and/or conformational characteristics of the receptor and
b) an extracellular planar potential-sensitive electrode wherein the cell is in operative contact with said electrode.
The device of the present invention comprises a living cell. This cell may be a microorganism, e.g. a bacterial cell or a yeast or fungal cell. Preferably, however, the cell is a eukaryotic cell, more preferably, a mammalian cell. Further, it is preferred that the cell overexpresses the ion channel/receptor system, i.e. the cell is manipulated, e.g. by genetic engineering or mutation in a way that components of the ion channel/receptor system are expressed in a higher amount than in a comparative untreated cell. More preferably, the cell is transfected with nucleic acid molecules encoding components of the ion channel/receptor system. In this embodiment of the invention the cell comprises heterologous nucleic molecules which encode at least a part of the components of the ion channel/receptor system and which allow overexpression of said components.
The ion channel/receptor system comprises a polypeptide or a plurality of polypeptides. On the one hand, the ion channel/receptor system comprises an ion channel component, e.g. a polypeptide or a plurality of polypeptides being capable of mediating an ion, i.e. cation and/or anion current through a cell membrane. On the other hand, the ion channel/receptor system comprises a receptor component which is responsive to stimuli. The receptor may be the ion channel or a part of the ion channel. The receptor, however, may be a molecule which is different from the ion channel, which is, however, in operative connection with the ion channel, e.g. a change in the functional and/or conformational state of the receptor results in a change of the functional state of the ion channel thus resulting in a detectable change of ion current through the cell membrane. The stimuli by which the receptor may be mediated are preferably selected from changes in the potential (inside or outside the cell), the presence or absence of effectors, e.g. ligands of the receptor, illumination, mechanical stimulation, stimulation by stimulation spots on the electrode or combinations thereof.
The cell is cultivated on a planar potential-sensitive electrode. Methods of cultivating cells on planar potential-sensitive electrodes are disclosed e.g. in S. Vassanelli, P. Fromherz xe2x80x9cNeurons from Rat Brain Coupled to Transistorsxe2x80x9d Appl. Phys. A 65, 85-88 (1997). By means of these cultivation cells are obtained, which grow on the potential-sensitive regions of the electrode resulting in an operative contact of the cell and the electrode.
The functional characteristics of the ion channels in the cell include an opening of the channels which will cause an ion current through all participating channels. These ion currents will also flow in the region of operative contact between cell and electrode resulting in a detectable signal which can be measured by the extracellular planar potential-sensitive electrode. The detectable signal may be e.g. a voltage drop due to a junction resistance by the narrow cleft between cell and substrate or the change of the surface potential of the electrode due to diffuse ion concentration changes in the operative contact zone.
A change in functional characteristics e.g. conductivity of the ion channel changes the ion current and therefore the electrical signal detected by the electrode. Since the ion channels are responsive to the effector-receptor system, an alteration in the effector-receptor system will modulate the opening of the ion channels and thus result in a detectable signal.
Ion channels, particularly the gating characteristics thereof, can be modulated by different methods, e.g. by voltage modulation across the membrane (voltage-gated ion channels), by ligands acting on the intracellular and/or extracellular side of the channel (ligand-gated ion channels), by mechanical changes (mechanically-gated ion channels) or by combinations thereof.
Voltage-gated ion channels, i.e. ion channels which are voltage sensitive, will change their conductivity with the potential drop over the membrane (Vm=Vintraxe2x88x92Vextra). If the electrolyte, i.e. the culture medium in which the cell is grown, is grounded (Vextra=0 mV) this potential drop equals the intracellular membrane (Vm=Vintra). This potential drop may be measured and/or modulated by patch clamp devices, i.e. electrodes which are inserted in or attached to the cell, and allow an adjustment of Vm to a fixed potential. In another embodiment, the conductivity of voltage-gated ion channels may be changed by voltage modulation due to an interaction with other ion channels, e.g. by means of an action potential. Vm is changed due to ion currents flowing into a cell through different ion channels. This co-operation of several ion channels influences the potential drop over the membrane leading in some cases to an action potential. Moreover, the potential difference between intracellular and extracellular side of the membrane may be modulated by using stimulation spots on the electrode.
A stimulation spot may be integrated next to the potential-sensitive electrode being in operative contact to the cell (Stett et al., Phys. Rev. E 55 (1997), 85). Thus, a device with the features of stimulation and recording may be built. A stimulation spot can, e.g. trigger an action potential which then will be recorded by the extracellular electrode.
Ligands can modulate ion channels preferably by two mechanisms, ionotropic and second messenger systems. In an ionotropic system the ligand molecules bind directly to the ion channels and alter their gating characteristics, e.g. intracellular Ca2+ shifts the gating curve of some K+ channels (DiChiara and Reinhard, J. Physiol. 489.2 (1995), 403). In second messenger systems the ligand molecules bind to a receptor which will first trigger some other molecules before the ion channel is influenced, e.g. many glutamate-second messenger systems.
Of course these different methods of modulating ion channels may be combined to create an effective biosensor which may be used for assaying the influence of a change in environmental parameters, e.g. a test substance (effector) on a receptor molecule in the cell.
The coupling of several ion channels may lead to a sudden and specific voltage drop over the membrane of the cell, called an action potential. To release an action potential there are at least two types of channels necessary, e.g. potassium and sodium channels. Both types of channels can be transfected into cells. Alternatively, one may use cell types which already have intrinsic receptors and respond with an action potential, e.g. chromaffin cells or nerve cells. To trigger an action potential one may use one or several of the techniques described above.
The combination of ion channels with the ability to release an action potential and a receptor-effector system is a powerful tool. The advantage of an action potential is the fast and large voltage drop over the membrane which can easily be detected with an extracellular electrode as an xe2x80x9ceventxe2x80x9d. If the signal is very weak, one may use techniques of averaging. Thus, a simple and uncomplicated device and method for assaying substances, if they influence the release of action potentials, is provided.
In a further embodiment of the invention caged probes may be used for a quick release of a large amount of a ligand. Biologically active substances, e.g. Ca2+ or the neurotransmitter L-glutamate may be released by UV-illumination, UV-lasers or flashlamps, and act on the receptor.
A specific example of a device according to the present invention are cells which are transfected with the xcex1- and xcex2-subunits of the voltage-dependent potassium channel hSlo. These cells are cultivated on a field-effect transistor. The characteristic gating curve of the ion channel may be shifted by xcex2-estradiol (Valverde et al., Science 285 (1999), 1929). This shift corresponds to the opening of ion channels. By changing the extracellular concentration of xcex2-estradiol an ion current will flow and can be detected by the field-effect-transistor under the cells. This system may be used as a sensor for xcex2-estradiol concentration.
In another preferred embodiment cells are transfected with a nucleic acid encoding an ionotropic receptor for glutamate, e.g. the NMDA receptor. The ionic flux through the receptor consists of potassium and sodium ions. This ionic current may be triggered by extracellular glutamate addition and recorded by an extracellular electrode. Thus, this system may be used as a sensor for gluatmate concentration. Some characteristics of NMDA channels show the high suitability to use this channels as a part of a sensor (single channel conductivity 50 pS; selectivity for cations: K+, Na+ and Ca2+; voltage dependency; channel will only open in the presence of glycine; Kandel et al., Neurowissenschaften 236 (1996), Spektrum Verlag).
In an embodiment of the invention a cell which is transfected with receptors for L-glutamate is grown on a chip. A certain amount of L-glutamate is released by flashlight. By this means, the receptor will be opened and an ion current begins to flow. The ion flow may be detected by the electrode. This means that the ion current through the receptor may be triggered by a flashlight.
When a cell is attached to the electrode surface, which may be oxidized silicon, other insulated semiconductors or metal, the cell membrane and the electrode surface are separated by a cleft which may be filled with an electrolyte as illustrated in FIG. 1. Thus, a sandwich structure is formed of e.g. silicon, silicon dioxide, cleft, cell membrane and cell interior. The electrode may be integrated on, e.g. embedded in a chip. The chip may comprise further devices such as stimulating spots, transistors etc. Preferably the chip has at least one integrated field-effect transistor comprising at least one source and drain or an electrode as stimulating spot for applying voltages. The potential sensitive electrode, however, may also be a metal electrode which may be integrated on a chip.
The equivalent circuit of the planar core-coat conductor in a cell-silicon junction is shown in FIG. 1a+b. Capacitances are assigned to the membrane and to the oxide in the junction. One or several ion conductances in the attached membrane may be driven by Nernst potentials. The extended cleft is represented by an ohmic conductance. The free part of the cell is described by a capacitance and one or several ion conductances, too. The ionic and capacitive currents in the circuit determine the intracellular voltage VM and the extracellular voltage VJ in the cleft. The voltage VJ in the junction controls the transistor. It plays the same role as the gate-voltage on the metalized gate of a common MOS-FET.
We apply Kirchhoff""s law to the node in the junction of FIG. 1b, and obtain Eq. 1 for the voltage VJ, with the membrane capacitance per unit area cM, the ion conductance g1JM per unit area of the membrane in the junction, the reversal voltage V10 and a cleft conductance gJ per unit area of the junction. The approximation of Eq. 1 is valid for weak coupling, i.e. for small values of VJ and dVJ/dt at a modest electrode capacitance cOX.                                           g            J                    ⁢                      V            J                          =                                            g              JM              1                        ⁡                          (                                                V                  M                                -                                  V                  0                  1                                            )                                +                                    c              M                        ⁢                                          ⅆ                                  V                  M                                                            ⅆ                t                                                                        (        1        )            
The properties of the planar core-coat conductor are xe2x80x9csqueezedxe2x80x9d into the cleft conductance gJ per unit area of the junction according to Eq. 2 with the distance dJ of membrane and substrate, with the specific resistance xcfx81J of the electrolyte in the cleft and with the radius aJ of a circular junction.                               g          J                =                                            5              ⁢                              xe2x80x83                            ⁢              π              ⁢                              xe2x80x83                            ⁢                              d                J                                                    ρ              J                                ⁢                      xe2x80x83                    ⁢                      1                          π              ⁢                              xe2x80x83                            ⁢                              a                J                2                                                                        (        2        )            
We may eliminate the capacitive current in Eq. 1 by taking into account Kirchhoff""s law for the intracellular node of FIG. 1b. The capacitive current through the total membrane is balanced by the total ion current through the free and the attached areas of the membrane according to Eq. 3, with the specific conductances g1FM and g1JM in the two regions and with the ratio xcex2 of the areas of attached and free membrane. Again the approximation of Eq. 3 is valid for weak coupling, i.e. for small values of VJ and dVJ/dt.                                           (                          1              +              β                        )                    ⁢                      xe2x80x83                    ⁢                      c            M                    ⁢                                    ⅆ                              V                M                                                    ⅆ              t                                      =                              -                          (                                                g                                      F                    ⁢                                          xe2x80x83                                        ⁢                    M                                    1                                +                                  β                  ⁢                                      xe2x80x83                                    ⁢                                      g                    JM                    1                                                              )                                ⁢                      (                                          V                M                            -                              V                0                1                                      )                                              (        3        )            
Inserting Eq. 3 into Eq. 1, taking into account Eq. 2, we obtain the coupling relation for an ionoelectronic sensor according to Eq. 4.                               V          J                =                                                            ρ                J                            ⁢                              a                J                2                                                    5              ⁢                              xe2x80x83                            ⁢                              d                J                                              ⁢                      xe2x80x83                    ⁢                                                    g                                  J                  ⁢                                      xe2x80x83                                    ⁢                  M                                1                            -                              g                                  F                  ⁢                                      xe2x80x83                                    ⁢                  M                                1                                                    1              +              β                                ⁢                      (                                          V                M                            -                              V                0                1                                      )                                              (        4        )            
The relation shows that a large signal on the gate requires:
(i) a small distance dJ of membrane and substrate,
(ii) a large radius aJ of the contact,
(iii) an enhanced or depleted conductance of the receptor channels in the attached membrane with g1JMxe2x88x92g1FMxe2x89xa00, and
(iv) an electrochemical driving force VMxe2x88x92V10.
Thus, the bioelectronic device of the invention is suitable as a sensor which allows the determination of a change in an environmental parameter as a detectable signal on the electrode and which is suitable as a scientific tool for studying the conformational and functional states of membrane proteins.
Particularly, the environmental parameter is an effector for the receptor component of the ion channel/receptor system. More particulary, the system is used to determine, if a test substance is capable of activating or inhibiting the receptor component of the ion channel/receptor system. The receptor component may be a pharmaceutically relevant target molecule. Thus, the present invention provides a method for contacting a test substance with a bioelectronic device as described above, wherein said bioelectronic device comprises a cell expressing and preferably overexpressing an ion channel/receptor system, wherein a response of the receptor to the test substance is determined by an electric signal in the electrode of the bioelectronic device.
In another embodiment, the bioelectronic device may be used as a sensor to determine the presence or the amount of a substance which acts as an effector to the receptor component of the bioelectronic device.